Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus

ABSTRACT The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited. IMPORTANCE The conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.


Fitness of rusT mutants.
Efficiency of plating of C. crescentus wild-type, ΔrusT or RusT overexpressing (pP van -RusT in ΔvanAB) cells on PYE plates after 7 h of growth in PYE or PYE supplemented with the indicated concentration of ZnSO 4 or DIP, respectively.For RusT overexpression, cultures of ΔvanAB cells carrying pP van -RusT were additionally supplemented with 0.5 mM vanillate.Spots are 10-fold serial dilutions starting from OD 660 of 0.2.
To analyse RNA stability, cells grown in M2G were treated with rifampicin (200 µg/mL) at an OD 660 of 0.5 to terminate transcription.RNA samples were collected at the indicated time points and transcript levels were determined by Northern Blot analysis.Escherichia coli strains were grown aerobically at 37 °C in LB broth and supplemented with kanamycin (50 µg/mL), chloramphenicol (20 µg/mL) or tetracyclin (12 µg/ml) where appropriate.For conjugation, E. coli WM3064 was grown in LB with antibiotics at 30 °C under agitation in media supplemented with 0.3 mM meso-diaminopimelic acid (mDAP) to enable growth for WM3064 derivatives (6).

Hfq co-IP
Duplicates of C. crescentus wild-type and cells expressing 3XFLAG-Hfq (KFS-0344) were grown in PYE medium to OD 660 of 1. Expression of the tagged protein under the tested condition was confirmed by immunoblot analysis (Fig. S1B).Cell pellets corresponding to 50 OD 660 were collected and subjected to immunoprecipitation as described previously (7).cDNA libraries were prepared using the NEBNext Small RNA Library Prep Set for Illumina (NEB; E7300) according to the manufacturer's instructions.cDNA libraries were pooled and sequenced using an Illumina MiSeq system in paired end mode.Demultiplexed raw reads were imported into the CLC Genomics Workbench (Qiagen) and subjected to quality control and adaptor trimming.The trimmed reads were mapped to the Caulobacter crescentus NA1000 reference genome (NC_011916) with standard parameter settings.The dataset has been deposited at the National Center for Biotechnology Information's Gene Expression Omnibus (GEO) repository (8), and is available via the GEO accession GSE148206.
Transcriptome analysis using RNA-seq C. crescentus (ΔvanAB) cells carrying either the control vector pBV-MCS6 or a plasmid to express RusT under control of the vanillate-inducible promoter (pVan-RusT; pKF482-1) were grown in triplicates in M2 medium supplemented with glucose.RNA samples were collected prior to and 15 min after addition of vanillate to the culture at OD 660 of 0.8.Total RNA was purified, digested with DNase I, and RNA integrity was confirmed using a Bioanalyzer (Agilent).cDNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, #E7760) and sequenced using a HiSeq 1500 System in single-read mode.The read files in FASTQ format were imported into CLC Genomics Workbench (Qiagen), trimmed and mapped to the Caulobacter crescentus NA1000 reference genome (NC_011916) using the "RNA-Seq Analysis" tool with standard parameters.Read counts were normalized (CPM) and transformed (log 2 ).Differential expression was tested using the built in tool corresponding to edgeR in exact mode with tagwise dispersions.Genes with a fold change ≥ 2.0 and a FDRadjusted p-value ≤ 0.05 were considered as differentially expressed.The dataset has been deposited at the GEO repository, and is available via the GEO accession GSE148208.

Chromatin Immunoprecipitation coupled to deep sequencing (ChIP-seq)
Culture of exponentially growing C. crescentus CB15 ntrX::ntrX-HA (OD 660 of 0.5, 80 ml of culture in PYE) was supplemented with 10 μM sodium phosphate buffer (pH 7.6) and then treated with formaldehyde (1% final concentration) at RT for 10 min to achieve crosslinking.Subsequently, the culture was incubated for an additional 30 min on ice and washed three times in phosphate buffered saline (PBS, pH 7.4).The resulting cell pellet was stored at -80 °C.After resuspension of the cells in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) containing 10 mM of DTT, the cell resuspension was incubated in the presence of Ready-Lyse lysozyme solution (Lucigen, #186002) for 10 min at 37 °C, according to the manufacturer's instructions.The lysate was sonicated (Bioruptor Pico) at 4 °C using 15 bursts of 30 sec to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 min at 4 °C.The volume of the lysate was then adjusted to 1 ml using ChIP buffer (0.01% SDS, 1.1% Triton X-84 100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl) containing protease inhibitors (Roche) and pre-cleared with 80 μl of Protein-A agarose (Roche) and 100 μg BSA.Five percent of the pre-cleared lysate was kept as total input sample (negative ChIP control sample).The rest of the pre-cleared lysate was then incubated overnight at 4°C with a monoclonal rabbit Anti-HA Tag antibody (Millipore, clone 114-2C-7; 1:400).The immuno-complexes were captured by incubation with Protein-A agarose beads (pre-saturated with BSA) during a 2 h incubation at 4°C and then, washed subsequently with low salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), with high salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), with LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and finally twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).The immuno-complexes were eluted from the Protein-A beads with two times 250 μL elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, just like the total input sample, incubated overnight with 300 mM NaCl at 65°C to reverse the crosslinks.The samples were then treated with 2 μg of Proteinase K for 2 h at 45°C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5).DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), ethanol-precipitated using 20 μg of glycogen as a carrier and resuspended in 30 μl of DNase/RNase free water.
Immunoprecipitated chromatins were used to prepare sample libraries used for deepsequencing at Fasteris SA (Geneva, Switzerland).ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following the manufacturers' instructions.A single-end run was performed on an Illumina Next-Generation DNA sequencing instrument (NextSeq High), 50 cycles were performed and yielded several million reads per sequenced samples.The singleend sequence reads stored in FastQ files were mapped against the genome of C. crescentus NA1000 (NC_011916.1) using Bowtie2 Version 2.4.5+galaxy1available on the web-based analysis platform Galaxy (https://usegalaxy.org) to generate the standard genomic position format files (BAM).ChIP-Seq reads sequencing and alignment statistics are summarized in Supplementary Table S2.Then, BAM files were imported into SeqMonk version 1.47.2 (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) to build ChIP-Seq normalized sequence read profiles.Briefly, the genome was subdivided into 50 bp, and for every probe, we calculated the number of reads per probe as a function of the total number of reads (per million, using the Read Count Quantitation option).Analysed data illustrated in Figure 4 are provided in Supplementary Table S2.Using the web-based analysis platform Galaxy (https://usegalaxy.org),NtrX-HA ChIP-Seq peaks were called using MACS2 Version 2.2.7.1+galaxy0 (No broad regions option) relative to the total input DNA samples.The q-value (false discovery rate, FDR) cut-off for called peaks was 0.05.Peaks were rank-ordered according to their fold-enrichment values (Supplementary Table S2, peaks with a foldenrichment values >2 were retained for further analysis).Sequence data have been deposited to the Gene Expression Omnibus (GEO) database (GSE247928 series, accession numbers GSM7903192 and GSM7903193).

Primer extension analysis
For primer extension, 5 μg of RNA were denaturated in the presence of 1 pmol 5′ end-labelled primer (KFO-0966) at 70°C for 2 min and adjacently chilled on ice for 5 min.Next, the samples were mixed with the reaction mix (1X first strand buffer, 5 mM DTT, 0.5 mM each dATP, dGTP, dCTP and dTTP) at 42°C, and SuperScript III (100 U; Invitrogen) was added.cDNA synthesis was performed at 50°C for 60 min, followed by incubation at 70°C for 15 min to inactivate the enzyme.Samples were treated with RNase H (2.5 U) for 15 min at 37°C and the reaction was stopped by the addition of GLII loading buffer.Samples was separated electrophoretically together with a template-specific ladder (prepared using the SequiTherm EXCELII DNA Sequencing Kit) on a 6% sequencing gel.

Electrophoretic Mobility Shift Assays
Complex formation between sRNAs and Hfq was analysed in vitro using gel mobility shift assays following previously established protocols (11).In short, denatured 5' end labelled sRNA (4 nM final concentration) was incubated with purified C. crescentus Hfq (lab stock; concentration as indicated in the figure legend) in the presence of 1 µg yeast RNA and 1x structure buffer (0.01 M Tris-HCl [pH 7], 0.1 M KCl, 0.01 M MgCl2) or Hfq dilution buffer (1x structure buffer, 1% glycerol, 0.1% Triton X-100) at 30°C for 15 min.Reactions were mixed with native loading buffer (50% glycerol, 0.5x TBE, 0.2% bromophenol blue) and separated by native PAGE.Gels were dried and signals visualized on a phosphor imager.